universiti putra malaysia enhancement of …psasir.upm.edu.my/id/eprint/31933/1/fs 2012...

UNIVERSITI PUTRA MALAYSIA

ENHANCEMENT OF ENZYMATIC PROPERTIES OF T1 LIPASE BY SATURATION MUTAGENESIS AT GLUTAMINE 114

ROSWANIRA BINTI AB. WAHAB

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ENHANCEMENT OF ENZYMATIC PROPERTIES

OF T1 LIPASE BY SATURATION MUTAGENESIS

AT GLUTAMINE 114


DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2012

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ENHANCEMENT OF ENZYMATIC PROPERTIES OF T1 LIPASE BY

SATURATION MUTAGENESIS AT GLUTAMINE 114

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Doctor of Philosophy

July 2012

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To my loving husband, Fahrul Zaman bin Huyop and my four girls, Farhanah, Farhah,

Fathiah and Fatini and also my parents, Roslina binti Nik Musa and Abdul Wahab bin

Saad who have always been there for me.

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Abstract of the thesis presented to the Senate of the Universiti Putra Malaysia in

fulfillment of the requirement for the degree of Doctor of Philosophy

ENHANCEMENT OF ENZYMATIC PROPERTIES OF T1 LIPASE BY

SATURATION MUTAGENESIS AT GLUTAMINE 114

By


July 2012

Chairman: Professor Mahiran binti Basri, PhD

Faculty: Science

Rational design of the recombinant T1 lipase gene by saturation mutagenesis on the

oxyanion Gln114 was successfully carried out to afford a library of twenty lipase

variants. The objective of the study was to investigate the impact of a single point

saturation mutagenesis at the oxyanion Gln114 on the enzymatic behavior and

enantioselectivity of T1 lipase. Furthermore, the effect of such mutation in the active

site of T1 lipase has never been studied. The selection of the mutation site was based

on the close proximity of Gln114 residue to the catalytic machinery and is intimately

involved in the substrate-enzyme interaction. It was hypothesized that the mutation

could invoke substantial changes in the catalytic efficiency and enantioselectivity of T1

lipase.

Computational assessment using YASARA, FoldX and Voronoia 1.0, found significant

variations in potential energy, total cavity and protein compactness. It was anticipated

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that Q114L and Q114M are more stable than T1 lipase. Profiling of the seven enzyme

behaviors found Q114L to show a 10oC increase in optimum temperature as compared

to 70oC in T1 lipase. Amino acids of small side-chains, polar, acidic and basic,

generally, caused reduction in the optimum temperatures. A downward shift in pH

optimum from pH 9 to pH 8 was observed when non-polar and basic amino acids were

replaced into Gln114. pH optimum of lipases were unchanged upon substitution with

polar amino acids. Specificities towards hydrolysis of natural oils were also very

different from one lipase to another, with an overall reduction in lipase activity.

Various chemical reagents affected the lipase variants, namely, organic solvents,

surfactants, metal ions and inhibitors/chelating agents. Lipases substituted with non-

polar amino acids exhibited higher frequency of improved enzyme properties. Their

non-polar nature that does not interact strongly with charged molecules in the active

site could be the contributing factor. Less enzyme deactivation was observed in

hydrophobic solvents as compared to hydrophilic ones. This is due to the nature of

hydrophilic solvents that tend to partition preferentially into the cytoplasmic

membrane, while hydrophobic solvents tend to partition away. The enzymes showed

preference towards surfactant Span 20, KI and SC, as well as catalysis activation by

Sr(II). Substitution of Leu and Met into Gln114 resulted in variants Q114L and Q114M

that showed the most number of cumulative enhancements and were more competent

than T1 lipase.

In the esterification of menthol with butyric anhydride, T1 lipase afforded a higher

yield of 99.3% menthyl butyrate as compared to 75.1% in Q114L. The former gave

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optimum conditions at 60oC, 2.53 mg enzyme, molar ratio of butyric

anhydride:menthol at 1:1 to 1.5:1 and incubated for 13 h and 15 min, while the latter

were at 67.5oC, 3 mg enzyme, substrate molar ratio at 3.5:1 and incubated for 13 h and

21 min. The higher yield could be attributed to the hydrophilic nature of the substrates

that could interact better with the hydrophilic Gln114 in T1 lipase. Also, the higher

temperature in Q114L-catalyzed reactions could have increased evaporation of

menthol, hence, lowering its concentration in the reaction. In the enantioselectivity

study for the enzymatic resolution of racemic ibuprofen using oleyl alcohol, variants

Q114L and Q114M were selected for this purpose. Mutation at oxyanion Gln114 had

changed enantioselectivity, whereby Q114M was the most enantioselective followed

by T1 lipase and Q114L.

Hence, it can be concluded that saturation mutagenesis of oxyanion Gln114 had

significantly changed the enzymatic behavior and enantioselectivity of T1 lipase. The

observed cumulative improvement in lipase properties mostly came from substitutions

with mainly medium-sized non-polar hydrophobic amino acids. The most catalytically

competent variants were found to be Q114L and Q114M, respectively. On the other

hand, substitution with hydrophilic, basic, acidic and bulky side-chain amino acids

affected T1 lipase negatively. Hence, it is recommended that future mutational studies

to avoid using these amino acids as it would only lead to inferior enzyme qualities.

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Abstrak thesis dikemukakan ini kepada Senat Universiti Putra Malaysia bagi

memenuhi syarat untuk mendapatkan ijazah Doktor Falsafah

PENAMBAHBAIKAN CIRI-CIRI ENZIM T1 LIPASE MELALUI

MUTAGENESIS TEPU KE ATAS GLUTAMINA 114

Oleh

ROSWANIRA BINTI AB WAHAB

Julai 2012

Pengerusi: Profesor Mahiran binti Basri, PhD

Fakulti: Sains

Rekabentuk rasional ke atas gen rekombinan lipase T1 melalui mutagenesis tepu ke

atas oksianion Gln114 telah dilaksanakan untuk menghasilkan satu koleksi sebanyak

dua puluh varian lipase. Objektif kajian ini adalah untuk mengkaji kesan mutagenesis

tepu satu titik pada oksianion Gln114 terhadap sifat enzim dan enantioselektiviti lipase

T1. Tambahan pula, kajian ke atas kesan mutasi di tapak aktif lipase T1 masih belum

pernah dilakukan. Pemilihan lokasi mutasi adalah berdasarkan Gln114 yang terletak

berhampiran dengan tapak aktif dan terlibat dengan interaksi substrat-enzim.

Hipotesisnya adalah mutasi ini akan mencetuskan perubahan yang besar pada

kecekapan pemangkinan dan enantioselektiviti lipase T1.

Penilaian komputer menggunakan YASARA, FoldX dan Voronoia 1.0, menunjukkan

perubahan yang ketara dalam tenaga keupayaan, jumlah kaviti dan ketumpatan protein.

Dijangkakan Q114L dan Q114M akan lebih stabil berbanding lipase T1. Pemprofilan

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ke atas tujuh sifat enzim mendapati Q114L menunjukkan peningkatan sebanyak 10oC

suhu optimumnya berbanding 70oC pada lipase T1. Asid amino berantai sisi kecil,

berkutub, berasid dan berbes secara amnya, menurunkan suhu optimum. Peralihan

menurun pH optimum dari pH 9 hingga pH 8 berlaku apabila Gln114 digantikan

dengan asid amino tidak berkutub dan berbes. pH optimum lipase tidak berubah

apabila digantikan dengan asid amino berkutub. Kespesifikan hidrolisis terhadap

minyak semulajadi juga berubah dengan menunjukkan pengurangan secara keseluruhan

ke atas aktiviti lipase.

Pelbagai reagen kimia didapati memberi kesan ke atas varian lipase, iaitu, pelarut

organik, surfaktan, ion logam dan agen perencat/pengkelat. Lipase yang digantikan

dengan asid amino tidak berkutub menunjukkan kekerapan penambahbaikan sifat

enzim yang lebih `tinggi. Sifat asid amino tidak berkutub yang tidak berinteraksi kuat

dengan molekul di tapak aktif mungkin adalah faktor penyebab. Penyahaktifkan enzim

yang kurang di dalam pelarut hidrofobik berbanding hidrofilik mungkin disebabkan

oleh sifat pelarut hidrofilik yang cenderung untuk masuk ke dalam membran

sitoplasma, manakala pelarut hidrofobik pula berkecenderungan untuk berada di luar.

Sesetengah enzim menunjukkan kespesifikan terhadap surfaktan Span 20, KI dan SC

serta terhadap pengaktifan pemangkinan oleh Sr(II). Penggantian Leu dan Met ke atas

Gln114 menyebabkan varian Q114L dan Q114M menunjukkan penambahbaikan

terkumpul yang lebih tinggi dan pemangkinan yang lebih cekap daripada lipase T1.

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Pengesteran mentol dengan butirik anhidrida yang dimangkinkan lipase T1, keadaan

optimum adalah pada 60oC dengan 2.53 mg enzim, nisbah molar butirik

anhidrida:mentol adalah 1:1 kepada 1.5:1 dan 13 jam dan 15 minit pengeraman untuk

menghasilkan 99.3% mentil butirat. Bagi Q114L, keadaan optimum adalah 67.5oC, 3

mg enzim, nisbah molar butirik anhidrida:mentol sebanyak 3.5:1 dan dieram selama 13

jam dan 21 minit untuk hasil sebanyak 75.1%. Walaupun dalam keadaan optimum,

lipase T1 menghasilkan lebih banyak mentil butirat berbanding Q114L. Ini mungkin

disebabkan oleh sifat hidrofilik substrat yang berinteraksi lebih baik dengan Gln114

hidrofilik pada lipase T1. Suhu tindakbalas Q114L yang lebih tinggi juga mungkin

telah meninggikan kadar sejatan mentol dan menurunkan kepekatannya di dalam tindak

balas. Dalam kajian enantioselektiviti resolusi enzim ke atas ibuprofen rasemik

menggunakan alkohol olel, varian Q114L dan Q114M telah dipilih untuk tujuan ini.

Mutasi pada oksianion Gln114 telah mengubah enantioselektiviti lipase T1 di mana

Q114M merupakan yang paling enantioselektif diikuti oleh lipase T1 dan Q114L.

Kesimpulannya, mutagenesis tepu pada oksianion Gln114 telah mengubah sifat enzim

dan enantioselektiviti T1 lipase. Diperhatikan bahawa penambahbaikan terkumpul sifat

enzim secara amnya disebabkan oleh penukaran dengan amino asid hidrofobik tidak

berkutub dan yang bersaiz sederhana. Varian yang paling kompeten adalah Q114L dan

Q114M. Namun begitu, penukaran dengan amino asid hidrofilik, berbes, berasid and

berantai tepi yang besar memberi kesan negatif kepada T1 lipase. Dengan itu, untuk

kajian mutasi pada masa hadapan adalah disarankan penggunaan asid amino ini

dielakkan kerana ianya akan menghasilkan enzim yang berkualiti rendah.

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ACKNOWLEDGEMENTS

In the name of Allah S.W.T the compassionate and merciful, I would like to express

my deepest gratitude to Allah S.W.T. for allowing me to complete my study. I wish to

express my sincere appreciation to my supervisor, Prof. Dr. Mahiran Basri for her

valuable guidance, advice, supervision, patience, understanding and suggestions during

the period of this study. My appreciation also goes to my co-supervisors, committee

members and EMTECH group principle researchers; Prof. Dr. Raja Noor Zaliha Raja

Abdul Rahman, Dr. Adam Leow Thean Chor, Prof. Dr. Abu Bakar Salleh and Prof. Dr.

Mohd. Basyaruddin Abdul Rahman for their valuable comments, time, moral support

and encouragement.

I am also grateful to my Lab 140 members; Harmiza, Iffa, Ira, Fiza, Ferrol, Menega,

Zarir, Ely and so many others for their cooperation in one way or another. I would also

like to thank my labmates from EMTECH at the Institute of Bioscience and Chemistry

Laboratory 401 for their willingness to aid me during my work in their labs. I would

also like to thank Dr Naz Chaikshbash for taking her time and kindness she has shown

me. Finally, my deepest love and appreciation to my husband, Fahrul Zaman bin

Huyop. To my wonderful parents, Abdul Wahab bin Saad and Roslina binti Nik Musa,

who have always been there to for me. They left their home in Taiping to come live

with my entire family and have lovingly looked after them while I was away in

Serdang during the whole of my study period. I can never repay the so many sacrifices

that they have made for me and I will always be indebted to them.

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I certify that a Thesis Examination Committee has met on 13th

July 2012 to conduct the final examination of Roswanira binti Ab. Wahab on his (or her) thesis entitled "Enhancement of Enzymatic Properties of T1 lipase by Saturation

Mutagenesis at Glutamine 114" in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows: Zulkarnain Zainal, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Mohd Aspollah Hj Md Sukari, PhD

Professor

Faculty of Science


(Internal Examiner)

Suraini Abd Aziz, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences


(Internal Examiner)

Rickey Yoshio Yada, PhD

Professor

University of Guelph

Canada

(External Examiner)

SEOW HENG FONG, PhD

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia has been accepted

as fulfillment of the requirements for the Degree of Doctor of Philosophy. The

members of Supervisory Committee were as follows:

Mahiran Basri, PhD

Professor

Faculty of Science


(Chairperson)

Raja Noor Zaliha Raja Abdul Rahman, PhD

Professor



(Member)

Adam Leow Thean Chor, PhD

Senior Lecturer



(Member)

_______________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any

other institution.


Date: 13 July 2012

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I certify that a Thesis Examination Committee has met on 13th

July 2012 to conduct the final examination of Roswanira binti Ab. Wahab on his (or her) thesis entitled "Enhancement of Enzymatic Properties of T1 lipase by

Saturation Mutagenesis at Glutamine 114" in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows: Zulkarnain Zainal, PhD

Professor

Faculty of Science


(Chairman)

Mohd Aspollah Hj Md Sukari, PhD

Professor

Faculty of Science


(Internal Examiner)

Suraini Abd Aziz, PhD

Professor



(Internal Examiner)

Rickey Yoshio Yada, PhD

Professor

University of Guelph

Canada

(External Examiner)

SEOW HENG FONG, PhD

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL x

DECLARATION xii

LIST OF TABLES xiii

LIST OF FIGURES xv

LIST OF SCHEMES xix

LIST OF ABBREVIATIONS xx

CHAPTER

1 INTRODUCTION 1

Objectives

2 LITERATURE REVIEW

2.1 Redesigning Enzymes for Improvement in Enzymatic

Properties

4

2.2 Structure, Characteristics and the Molecular Mechanism of

T1 Lipase

5

2.2.1 Structural and Enzymatic Properties 5

2.2.2 The molecular mechanism of T1 lipase 6

2.3 The Oxyanion Hole in the α/β hydrolase Fold of T1 Lipase 7

2.4 Redesigning and Selection of Mutation Point in the T1

Lipase Protein

9

2.5 Saturation Mutagenesis of Enzymes 12

2.6 Reactions of Lipases 13

2.6.1 Hydrolysis 15

2.6.2 Esterification 15

2.7 Specificity of Lipases 19

2.7.1 Lipid Class Specificity 19

2.7.2 Fatty Acid Specificity 20

2.7.3 Regioselectivity 22

2.7.4 Stereospecificity 24

2.8 Application of Lipases 27

2.8.1 Synthesis of Chiral Drugs and Pharmaceutical

Intermediates

27

2.9 Enhancement of Lipase Catalytic Properties 29

2.9.1 Thermostability 29

2.9.2 Substrate Specificity 31

2.9.3 pH Stability 32

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2.9.4 Reaction in Organic Solvents 34

2.9.5 Addition of Surfactants 35

2.9.6 Addition of Metal Ions 37

2.9.7 Enantioselectivity 39

2.10 Factors that Affect Enantioselectivity of Lipases 41

2.10.1 Substrate Modification 41

2.10.2 Temperature 43

2.10.3 Organic solvents 44

2.10.4 pH 45

2.11 Determination of Enantioselectivity 47

2.12 Techniques in Customizing Lipases for Biocatalysis 48

2.12.1 Genetic Engineering 49

2.13 Genetic Modification of Enzyme 51

2.13.1 Polymerase Chain Reaction 50

2.14 Computer-aided rational design of proteins 52

2.14.1 FoldX 52

2.14.2 Voronoia 1.0 53

2.14.3 YASARA 54

3 MATERIALS AND METHODS

3.1 Materials 55

3.2 Computational Assessment and In-silico Studies 56

3.2.1 YASARA: In-silico Protein Mutation and

Visualization

56

3.2.2 FoldX: Calculation of Structural Stability 57

3.2.3 Voronoia 1.0: Calculation and Comparison of

Protein Compactness

57

3.3 Preparation of T1 Lipase Cultures 59

3.3.1 Preparation of Stock and Working Culture 59

3.3.2 Preparation of Plasmid 59

3.4 Site-directed Mutagenesis of T1 Lipase 60

3.4.1 Primer Design 60

3.4.2 Polymerase Chain Reaction 61

3.4.3 Transformation of PCR Amplified Mutated T1

Plasmid

61

3.4.4 Screening of Positive Recombinant Colonies 63

3.4.5 Extraction of Mutated Plasmid 63

3.4.6 Sequencing of T1 lipase and the 19 Lipase Variants 63

3.4.7 Lipase Activity and Protein Content 64

3.4.8 Partial Purification of Enzymes 64

3.5 Qualitative Determination of the Lipase Activity of T1

Lipase and the 19 Lipase Variants

65

3.5.1 Triolein Agar Plate 65

3.5.2 Tributyrin Agar Plate 66

3.5.3 Rhodamine Agar Plate

66

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3.6 Quantitative Determination of Lipase Activity and Protein

Content

66

3.6.1 Determination of Lipase Activity and Protein

Content

66

3.6.2 Analysis of Lipase Activity 68

3.7 Profiling the Characteristics of T1 lipase and the 19 Lipase

Variants

70

3.7.1 Optimum temperature 70

3.7.2 pH Optimum 71

3.7.3 Effect of Natural Oils 71

3.7.4 Effect of Organic Solvents 72

3.7.5 Effect of Surfactants 72

3.7.6 Effect of Metal Ions 73

3.7.7 Effect of Chelating Agents and Inhibitors 73

3.8 Calculation of Residual Activity 74

3.9 Esterification of Menthol with Butyric Anhdyride Catalyzed

by T1 lipase and Q114L

74

3.9.1 Effect of Reaction Conditions 75

3.10 Optimization of Esterification of Menthol with Butyric

anhydride Using Surface Response Methodology

76

3.10.1 Experimental design, and Optimization for Wild

type (WT) and Q114L Lipase

76

3.11 Statistical Analysis For The Response Surface Methodology

for the Optimization of Menthol with Butyric Anhydride

79

3.12 Enantioselective Esterification of (R-S)-ibuprofen with Oleyl

Alcohol

80

3.12.1 Effect of Reaction Conditions 80

3.13 Statistical Analysis 83

4 RESULTS AN DISCUSSION 85

4.1 Computational Assessments and In-silico Protein Studies 85

4.1.1 YASARA: In-silico Construction of Lipase

Variants and Visualization

85

4.1.2 FoldX: Calculation of Structural Stability 87

4.1.3 Voronoia 1.0: Calculation and Comparison of

Protein Compactness

88

4.2 Site-directed Mutagenesis of T1 lipase to Generate Lipase

Library

92

4.2.1 Classification of Lipase Variants 92

4.2.2 Screening of Positive Recombinant Colonies 92

4.3 Qualitative Determination 95

4.3.1 Lipase Activity 95

4.4 Quantitative Determination of Lipase Variants 97

4.4.1 Lipase Activity and Protein Content 97

4.5 Partial purification of Lipases 97

4.5.1 Preparation of Partially Purified Lipases 97

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4.6 Profiling the Characteristics of the Lipase Variants of

Recombinant T1 lipase

98

4.6.1 Statistical Analysis 98

4.6.2 Optimum Temperature 101

4.6.3 pH Optimum 105

4.6.4 Effect of Various Natural Oils 109

4.6.5 Effect of Organic Solvents 116

4.6.6 Effect of Surfactants 123

4.6.7 Effect of Metal Ions 129

4.6.8 Effect of Chelating Agents and Inhibitors 135

4.7 Esterification of Menthol with Butyric Anhydride 141

4.7 Effect of Incubation time 141

4.7 Effect of temperature 143

4.7 Effect of enzyme amount 145

4.7 Effect of substrate molar ratio 147

4.7 Effect of agitation speed 149

4.8 Optimization of the Esterification of Menthol with Butyric

Anhydride Catalyzed by the Wild-type T1 lipase Using

Response Surface Methodology

151

4.8.1 Model Fitting and Analysis of Variance (ANOVA) 151

4.8.2 Mutual effects of factors on the conversion of

menthol

157

4.8.3 Attaining optimum conditions and verification of

model

165

4.9 Optimization of the Esterification of Menthol with Butyric

Anhydride by Catalyzed by Q114L Using Response Surface

Methodology

165

4.9.1 Model Fitting and Analysis of Variance (ANOVA) 165

4.9.2 Mutual effects of factors on the conversion of

menthol

170

4.9.3 Attaining optimum conditions and verification of

model

176

4.9.4 Reaction Mechanism for the Esterification of

Menthol with Oleyl Alcohol

179

4.9.5 Summary of the Optimization Studies Between T1

lipase and Q114L

181

4.10 Enantioselective Esterification of (R-S)-ibuprofen with Oleyl

Alcohol

182

4.10.1 Statistical Analysis 182

4.10.2 Effect of Incubation time 182

4.10.3 Effect of Temperature 186

4.10.4 Effect of Enzyme Loading 190

4.10.5 Effect of Substrate Molar Ratio 193

4.10.6 Effect of Solvents 197

4.10.7 Effect of the Presence of Dessicant 202

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4.10.8 Reaction Mechanism for the Esterification of (R,S)-

ibuprofen with Oleyl Alcohol

206

5 CONCLUSIONS 209

5.1 Future Recommendations 212

REFERENCES

APPENDICES

BIODATA OF STUDENT

213

253

311

PUBLISHED PAPERS 313

universiti putra malaysia enhancement of …psasir.upm.edu.my/id/eprint/31933/1/fs 2012...

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